Mosquito-borne viruses, or arboviruses, are among the most important emerging pathogens worldwide with a significant potential impact on human health, and are also prominent components of the NIAID Category A, B, and C priority pathogens lists. The inclusion of many arboviruses as high priority pathogens is due in part to their virulence, the potential for vector-mediated dissemination, and the public concern regarding insect-borne viral infections. In addition, despite the worldwide impact of arboviruses, few effective treatments are currently available. The objective of this proposal is to use western equine encephalitis virus (WEEV), a category B arbovirus, to identify and develop novel antiviral compounds isolated from natural product extracts produced by marine sediment-derived microbes. Compounds derived from terrestrial and marine microorganisms have historically provided a rich source for novel therapeutics against a range of microorganisms, but have thus far been underutilized in the development of antiviral agents. We have already developed, validated, and employed a WEEV replicon-based assay to complete high-throughput screens against a library of >16,000 pre- fractionated extracts derived from a diverse array of marine microbial species. Furthermore, we have completed both analytical and preparative preliminary chromatographic fractionation procedures for two distinct extracts with validated antiviral activity. The specific aims of this proposal are designed to complete the isolation and functional characterization of active compounds from these two candidate extracts, including structural examination and initial viral target identification, and to expand the identification of microbe-derived natural products with anti-arboviral activity. The long term goals of this research project are to develop a wide range of antiviral compounds derived from marine microbes, characterize their structures and antiviral activities, and complete their preclinical testing in animal models of arbovirus-mediated disease, with the ultimate objective being clinical implementation of these candidate novel drugs. The experiments outlined in this exploratory project proposal will be used to establish the groundwork to accomplish these goals.